From 2006.igem.org

Revision as of 21:25, 15 May 2006

Name: Fousion

News

• 2006-05-15 Lab Notebook page created
• 2006-04-14 Discussion page created
• 2006-03-16 First kick-off meeting and team selection

If there is a specific task that we need the bacteria to do (e.g. change colour on cue, aggregate...) list it here. We have a TON of composite bricks, and can plan custom ones. BUT I need to know what U want... I am going nuts looking at the list

Your Dyslexic Dinosaur

Organisation

People

Students

Supervisors

Timeline

• Meetings
• Lab Notebook

Tasks

• Task Assignment

Modelling people: check out these bricks: BBa_I13920, BBa_I13921, BBa_I13922. What happens if you plug these into your computer simulation?

Also try this: BBa_J11002 and BBa_J06913. What happens if you substitute an AAV-degradation-tagged GFP?

Test EcoMscL to see if it does anything cool How many vials of competent Mscl(-) E. coli do we have? *make more* Do we have a luciferase reporter? What is its selection marker Amplify the Eco MscL plasmid

Useful papers

A synthetic gene–metabolic oscillator Eileen Fung1,2, Wilson W. Wong1, Jason K. Suen1, Thomas Bulter1, Sun-gu Lee1 & James C. Liao1,2 NATURE | VOL 435 | 5 MAY 2005 http://www.nature.com/nature/journal/v435/n7038/abs/nature03508.html

Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):679-84. Synchronizing genetic relaxation oscillators by intercell signaling. McMillen D, Kopell N, Hasty J, Collins JJ. http://www.pnas.org/cgi/content/full/99/2/679

Cell Mol Life Sci. 2006 May The design of intracellular oscillators that interact with metabolism. Wong WW, Liao JC.

Parts shopping list

Composite bricks - these are the best to use as less cloning is required.

All these bricks express YFP and downregulate it in response to a signal

YELLOW

ARABINOSE RESPONSE.

BBa_E0610: Arabinose turns on lac repressor which in turn shuts down Yellow fluorescent protein.

BBa_E0600: like BBa_E0610 but YFP is destabilized - slow degradation. Not listed as available.

BBa_E0611: Arabinose turns on destabilized TetR repressor which shuts down yellow fluorescent protein. Addition of Tetracyline blocks TetR repressor allowing yellow fluorescent protein to be turned back on.

BBa_E0601: Like BBa_E0611 but YFP is destabilized - slow degradation. Not listed as available

BBa_E0613: Arabinose turns on destabilized lambda repressor which shuts down yellow fluorescent protein.

BBa_E0602: Like BBa_E0613 but YFP is destabilized - slow degradation. Not listed as available

BBa_E0603: Like BBa_E0602 but baseline expression of YFP is higher. Not listed as available

BBa_E0604: Arabinose turns on destabilized 434 cl epressor which in turn shuts down destabilized (slow) Yellow fluorescent protein. Not listed as available

BBa_E0614: Like BBa_E0604 but YFP is not destabilized (hangs around forever). Not listed as available

BBa_E0605: Arabinose turns on destabilized p22 repressor which in turn shuts down destabilized (slow) Yellow fluorescent protein. Not listed as available

BBa_E0615: Like BBa_E0605, but YFP is not destabilized, hangs around forever.

Lac I REGULATED

BBa_I12026: Produces YFP (not destabilized). Controlled by Lac I BBa_I14022: Slow-destabilized (LVA) YFP controlled by Lac promoter

MULTIPLE REGULATORS

BBa_I12051: Lactose induces lambda repressor. Arabinose induces 434 repressor. Lambda repressor activates YFP, 434 represses YFP. YFP is destabilized (AAV, fast).

BBa_I12052: Like BBa_I12051 but promoter for YFP has double binding sites for better control.

LAMBDA regulated

BBa_I13973: Fast destabilized (AAV) YFP controlled by original lambda promoter - lambda protein would repress it.

LAS regulated

BBa_I14013: Slow destabilized YFP under Las promoter.

BBa_I14030: TetR promoter controls LacI repressor, lambda repressor, and YFP. All three are slow (LVA) destabilized.

BASIC COLOR LEGO

BBa_I14005: Slow-destabilized (LVA) YFP with strong ribosome binding site

CYAN

TET response

BBa_I0401: Expresses CFP and lux from the Tet-OFF promoter. Neither is destabilized. Not listed as available

BBa_I0402: Like BBa_I0401 but lux is destabilized (LVA, slow)

BBa_I0404: Produces rhlR and CFP, downregulated by Tet. rhlR binds with N-butyryl-HSL, regulates transcription. rhlR but not CFP is destabilized.

BBa_I0405: same as BBa_I0404, but produces rhIl which produces N-butyryl-HSL.

BBa_I0406: produces CFP and lasR, which accepts chemical signal AI-1. Downregulated by Tet. LasR but not CFP are destabilized.

BBa_I0407. Like BBa_I0406 but produces lasI (destabilized LVA slow) which generates the chemical signal Al-1.

BBa_I0408: produces CFP and CinR, repressed by Tetracycline. Cin R is LVA (slow) destabilized. Cin R binds O3-C14:1-HSL and activates the Cin promoter.

BBa_I0409: Like BBa_I0408 but produces cinL (destabilized LVA slow) which generates the chemical signal O3-C14:1-HSL.

BBa_I14029: LacI promoter controls slow destabilized (LVA) TetR and slow destabilized (LVA) YFP

ARABINOSE response:

BBa_I0403 Expresses CFP and aiiA from an arabinose promoter. aiiA degrades N-acyl homoserine lactones (quorum sensing autoinducers). aiiA but not CFP is destabilized (LVA, slow).

GREEN

ARABINOSE

BBa_I13540: Arabinose turns on green fluorescent protein.

RED:

BBa_I13507: Basic red fluorescent protein: ribosome binding site, red, stop. B.Y.O. promoter.

ARABINOSE

BBa_I13520: Arabinose turns on mRFP. This think was actually shown to work. BBa_I13517: Like BBa_I13520 but weaker ribosome binding site.

SIGNALING

Promoterless (BYOP)

BBa_I0460 aiiA cassette. Produces enzyme which degrades N-acyl homoserine lactones (quorum sensing autoinducers. Destabilized with LVA (slow) B.Y.O.P (Bring your own promoter).

BBa_I10100: Produces adhesin, makes bacteria stick to each other. B.Y.O.P (Bring your own promoter)

BBa_I14002: LVA-destabilized Lac I, with strong ribosome binding site.

BBa_I14003: LVA-destabilized TetR, with strong ribosome binding site.

BBa_I14010: LVA-destabilized Ara C, with strong ribosome binding site.

BBa_I14012: LVA-destabilized LasR, with strong ribosome binding site.

BBa_I14045: LVA-destabilized RhiR, with strong ribosome binding site

BBa_I14046: LVA-destabilized RhiR, with strong ribosome binding site

With promoter

BBa_I14023: Constitutively expresses slow-destabilized LasR protein. BBa_I14023: Constitutively expresses slow-destabilized RhiR protein.

BBa_I12002: Lambda promoter driving expression of 434 repressor. Not destabilized. BBa_I12054: same as BBa_I12002, lambda promoter is modified to be activated by lac repressor cI

BBa_I12055: Hybrid lambda promoter activated by lambda and repressed by 434, driving expression of destablized lambda (LVA slow degradation). This was used as part of an oscillator. BBa_I12056: Like BBa_I12055:but the promoter has duplicate sites for better control.

BBa_I12011: Lambda promoter driving expression of p22 repressor. LVA (slow) destabilized.

BBa_I13271: Cassette: rhiR upstream of rhi promoter. rhiR with butyl-homoserine lactone activates rhi promoter. rhiR needs a promoter, not included. BBa_I13303: Like BBa_I13271 but controlled by lac promoter. BBa_I13309: like BBa_I13303 BBa_I13310: like BBa_I13303 but controlled by TetR BBa_I13312: like BBa_I13303 but controlled by TetR

BBa_I14005, BBa_I14038: RhI promoter upstream of Lac I

BBa_I13305: Lac I induces LasR, LasR with homoserine lactone activates Las promoter (downstream). Can add your own gene after Las promoter.

BBa_I14014: slow destabilized (LVA) LasR under Lac I promoter

BBa_I13016, BBa_S04003: Arabinose induces LVA (slow) destabilized TetR protein.

BBa_I13017, BBa_S04002: Expression of LVA(slow) destabilized TetR protein controlled by lactose (Lac I promoter).

BBa_I14028, BBa_I14036: LVA-destabilized Lambda controlled by LasR promoter.

BBa_I14037: Destabilized LasL controlled by LasR promoter. Lambda operator allows shutdown by lambda repressor.

BBa_I14050: Destabilized RhiL upregulated by Las/HSL and blocked by TetT

BBa_J06204: Makes lambda cl protein in response to C4-HSL

CHAIN REACTION PREMADE:

BBa_I13036: Arabinose induces TetR. TetR induces LuxR. LuxR induces YFP if a homoserine lactone signal is received.

BBa_I13037: Lactose induces TetR. TetR induces LuxR. Arabinose induces LuxL which produces homoserine lactone. LuxR and homoserine lactone activate YFP.

BBa_I13038: Arabinose induces TetR, and also luxL which makes homoserine lactone. TetR induces LuxR. LuxL and LuxR together activate YFP.

BBa_I13920: Lux pL makes Lux R protein. Lux R protein + homoserine lactone shuts off Lux pL (no more lux R) and activates Lux pR which makes LacI. Lac I shuts off luxL protein (homoserine lactone) which shuts down lac I and HSL production, Lux pL is free to start over. Lac I and LuxL are slow destabilized, LVA.

BBa_I13921: Same as BBa_I13920 but TetR (LVA destabilized) is substituted for Lac I.

BBa_I13922: Same as BBa_I13920 but lambda repressor (LVA destabilized) is substituted for Lac I.

BBa_J06910: Lambda induces destabilized (slow LVA) LacI. LacI shuts down EYFP (nondestabilized).

BBa_J06913: If you add 434, you repress lambda (LVA slow destabilized). YFP is downstream of a lambda promoter so by adding 434 you reduce repression and turn on YFP.

MOTILITY

TetR control

BBa_I13710: TetR induces CheY, which is a phosphorylation target for motility.

BBa_I13711: TetR induces CheB. CheB can be phosphorylated and will demethylate Aspartate receptors.

BBa_I13712: TetR induces CheR. CheR opposes Che B.

LACTOSE control

BBa_I13721: Lactose induces CheB. (lac promoter)

MULTI-COMPONENT

BBa_I13730: Lac I controls Che B, TetR controls Che R. Che B opposes Che R (chemosensing)

BBa_M30109: light sensor. Activates OmpC and Represses Omp F promoter

BBa_J11002: Generates GFP pulse, slow, not destabilized.

BBa_J06917: Lambda induces 434, which represses YFP. not destabilized.

BASIC TOOLS

BBa_I13800: ampicillin resistance

BBa_I13801: kanamycin resistance

SIMPLE COLOUR BLOCKS

CYAN BBa_E0420 ECFP (RBS+ LVA- TERM): (B0034.E0020.B0015) BBa_I15012 ECFP (RBS+ LVA-): (B0030.E0020) BBa_I15016 ECFP (RBS+ LVA-): (B0032.E0020) BBa_I15019 ECFP (RBS+ LVA-): (B0031.E0020) BBa_I13600 ECFP (tetR promo RBS+ LVA- TERM): (R0040.E0420) BBa_I13601 ECFP (lacI/pL promo RBS+ LVA- TERM): (R0011.E0420) BBa_E0422 ECFP (RBS+ LVA+ TERM): (B0034.E0022.B0015) BBa_J04421 ECFP (LacI promo RBS+ LVA+ TERM): (R0010.E0422) BBa_I13602 ECFP (tetR promo RBS+ LVA+ TERM): (R0040.E0422), in absence of tetR ECFP is expressed BBa_I13603 ECFP (lacI/pL promo RBS+ LVA+ TERM): (R0011.E0422) BBa_E0024 ECFP (RBS- AAV+): more stable than E0022 but less than E0026? BBa_E0424 ECFP (RBS+ AAV+ TERM): (B0034.E0024.B0015) not listed as available BBa_E0026 ECFP (RBS- ASV+) BBa_E0426 ECFP (RBS+ ASV+ TERM): (B0034.E0026.B0015) not listed as available BBa_S03474: lambda represses CFP. Has RBS + TERM. Has double lambda sites. BBa_S03465: lambda activates CFP. Has RBS + TERM

YELLOW BBa_E0430 EYFP (RBS+ LVA- TERM): (B0034.E0030.B0015) BBa_I13001 EYFP (RBS+ LVA- TERM): (B0030.E0030.B0015) strong rbs BBa_I13002 EYFP (RBS+ LVA- TERM): (B0031.E0030.B0015) BBa_I13003 EYFP (RBS+ LVA- TERM): (B0032.E0030.B0015) BBa_I13004 EYFP (RBS+ LVA- TERM): (B0033.E0030.B0015) BBa_E0438 EYFP (RBS+ LVA+ TERM): (B0043.E0032.B0014): difficult to detect fluorescence unless under control of strong promoter at high copy BBa_J04671 EYFP (RBS- LVA+ TERM): (E0032.B0015) intermediate building sequence BBa_E0434 EYFP (RBS+ AAV+ TERM): (B0034.E0034.B0015) BBa_E0036 EYFP (RBS- ASV+): more stable than E0032 or E0034 BBa_E0436 EYFP (RBS+ ASV+ TERM): (B0034.E0036.B0015) not listed as available BBa_J03102: YFP can be turned on by lambda and off by 434. Too bad it is stable and not ASV/AAV tagged… BBa_J06005: YFP, repressed by lambda and turned on by LuxR + HSL BBa_J06105: YFP, repressed by lambda and turned on by LasR + HSL BBa_S03476: lambda represses YFP. Has RBS + TERM BBa_S03475: lambda represses YFP. Has RBS + TERM. Has double lambda sites. BBa_S03466: lambda activates YFP. Has RBS + TERM

GREEN BBa_E0040 GFP BBa_J04031 GFP (LVA+): (E0040 and LVA modeled on E0032), listed as M BBa_J04631 GFP (RBS- LVA+ TERM): (J04031.B0015), intermediate building sequence BBa_J04431 GFP (LacI promo RBS+ LVA+ TERM): (R0010.B0034.J04031.B0015) BBa_E0240 GFP (RBS+ TERM): (B0032.E0040.B0015) medium rbs BBa_E0241 : like BBa_E0240 but has a different terminator – BRICK SHOWN TO WORK! BBa_E0840 GFP (RBS+ TERM): (B0030.E0040.B0015) strong rbs BBa_J07034 GFP (FecA’ promo, RBS+ TERM): (J07018.E0840), not listed as available BBa_J07011 GFP (ctx promo, RBS+ TERM): (J07007.E0840), not listed as available BBa_I7102 GFP (tetR promo, sRBS, TERM): (R0040.B0030.E0040.B0015), not listed as available BBa_I13522 GFP (tetR promo RBS+ TERM): (R0040.B0034.E0040.B0015) BBa_J04430 GFP (LacI promo RBS+ TERM): (R0010.B0034.E0040.B0015) BBa_E0044 GFP (AAV+) BBa_E0244 GFP (RBS+ AAV+ TERM): (B0032.E0044.B0015) medium rbs, not listed as available BBa_E0844 GFP (RBS+ AAV+ TERM): (B0030.E0044.B0015) strong rb, not listed as available BBa_S03336: lambda activates GFP. Has RBS + TERM BBa_S03335: lambda suppresses GFP. Has RBS + TERM

RED BBa_E1010 mRFP1 (July 2004) BBa_I13521 mRFP1 (tetR promo RBS+ TERM): (R0040.B0034.E1010.B0015) BBa_J04051 mRFP1 (LVA+): listed as M BBa_J04651 mRFP1 (RBS- LVA+ TERM): (J04051.B0015) intermediate building sequence BBa_J04451 mRFP1 (LacI promo RBS+ LVA+ TERM): (R0010.B0034.J04051.B0015), not listed as available BBa_J04450 mRFP1 (LacI promo RBS+ TERM): (R0010.B0034.E1010.B0015) BBa_J06504: mCherry BBa_J06505: mCherry + LVA BBa_J06702: mCherry + RBS + TERM BBa_J06703: mCherry + RBS + LVA + TERM BBa_S03473: lambda represses RFP. Has RBS + TERM. Has double lambda sites. BBa_S03337: lambda activates RFP. Has RBS + TERM

COMPOSITE BBa_I15011 ECFP+EYFP: (B0030.E0020.B0030.E0030) single mRNA containing ECFP and EYFP produced under strong RBS BBa_I15015 ECFP+EYFP: (B0032.E0020.B0032.E0030) medium RBS BBa_I15018 ECFP+EYFP: (B0031.E0020.B0031.E0030) weak RBS BBa_J13003 ECFP+EYFP: (E0020.B0034.E0030.B0010.B0012): CFP produced in presence of RIPS (specific ribosome?), YFP produced in its absence

LacZ BBa_E0033 lacZ alpha fragment (RBS-): portion of LacZ gene

BBa_E0666 : Lac I controls CFP, Tet R controls YFP, lambda controls mRFP. None are destabilized. This could be a useful oscillator. Can we get this made with destabilized colours? BBa_E0669: as above, but lambda turns on yellow, LacI controls cyan, TetR controls red. BBa_I13604: Tet R controls yellow, LacI controls cyan, no degradation tags. BBa_I13605: Lac I controls yellow, TetR controls cyan, no degradation tags BBa_I13607: Lac I controls yellow, TetR controls cyan, LVA degradation tags.

Signaling promoters with colour

BBa_J07066, RFP controlled by FecA. Needs FecB BBa_J13000: construct BBa_J13020 : LasR + A1 signal induces YFP BBa_J13021: LuxR + HSL makes YFP BBa_J13025: LuxR + HSL makes CFP BBa_J13026: LuxR + HSL makes RFP

Bba_M30000: LacZ controlled by osmosensor

SINGLE BLOCKS - PROMOTERS

BBa_I14018 P(Bla) , constitutive, medium BBa_I14033 P(Cat), constitutive medium BBa_I14034 P(Kat), constitutive medium, BBa_I14032 P(Lac) IQ, constitutive strong BBa_I0500 acrtivated by arabinose BBa_J03007 activated by maltose BBa_R0010 repressed by lac I BBa_R0011 activated by lambda, repressed by lac I BBa_R0051 repressed by lambda BBa_R1051 repressed by lambda BBa_I12007 Modified lambda Prm promoter (OR-3 obliterated), activated by lambda BBa_I12006 Modified lamdba Prm promoter (repressed by 434 cI, activated by lambda) BBa_I12036 Modified lamdba Prm promoter (cooperative repression by 434 cI, activation by lambda) – dual sites for better control BBa_I12010 Modified lamdba Prm promoter (repressed by p22 cII) BBa_R0053 reprressed by p22 BBa_R1053 reprressed by p22 BBa_R1052 repressed by 434 BBa_R0040 (tetR, negative) BBa_I1051 Lux cassette right promoter (needs HSL) BBa_R0061 Lux R + HSL represses BBa_R0062 Lux R + HSL activates BBa_R1062 Lux R + HSL activates BBa_R0063 (luxR & HSL regulated -- lux pL). Weak constitutive, shut down by Lux + HSL BBa_R0065 activated by Lux + HSL, repressed by lambda BBa_I14017 P(Rhl), needs HSL BBa_R0071 activated by RhlR + C4-HSL BBa_R0079 activated by LasR + HSL BBa_R0074 repressed by pen I

SINGLE BLOCKS - RIBOSOME BINDING SITES

BBa_B0030 RBS.1 (strong) BBa_B0031 RBS.2 (weak) BBa_B0032 RBS.3 (medium) BBa_B0033 RBS.4 (weakest) BBa_B0034 RBS (strongest)

SINGLE BLOCKS - TERMINATORS

BBa_B0010 Terminator Forward BBa_B0011 Terminator Bidirectional BBa_B0014 Terminator Forward BBa_B0015 Terminator Forward BBa_B0021 Terminator Bidirectional BBa_B0025 Terminator Reverse

SINGLE BLOCKS - ACTIVATORS, REPRESSORS

BBa_C0012 Repressor, lacI (LVA+) BBa_C0040 Repressor, tetR (LVA+) BBa_C0050 Repressor, HK022 cI (RBS- LVA+) BBa_C0051 Repressor, Lambda cI (RBS- LVA+) BBa_C0052 Repressor, 434 cI (RBS- LVA+) BBa_C0053 Repressor, P22 c2 (RBS- LVA+) BBa_C0062 Repressor/Activator, luxR BBa_C0071 Repressor/Activator, rhlR (LVA+) BBa_C0072 Repressor, mnt (strong) (LVA+) BBa_C0073 Repressor, mnt (weak) (LVA+) BBa_C0074 Repressor, penI (LVA+) BBa_C0171 repressor rhlR (-LVA) BBa_C0177 activator cinR (-LVA) BBa_C0077 Activator, cinR (LVA+) BBa_C0079 Activator, lasR (LVA+) BBa_C0179 represspor lasR (-LVA) BBa_C0080 Repressor/Activator, araC (LVA+) BBa_C0024 CheB, demethylates aspartate receptors (for chemotaxis) BBa_C0028 CheR coding region, methylates aspartate receptors (for chemotaxis)

SINGLE BLOCKS - ENZYMES

BBa_C0060 Enzyme, aiiA (LVA+), Makes signaling molecule BBa_C0160 Enzyme aiiA (-LVA) BBa_C0061 Enzyme, luxI (LVA+), Makes signaling molecule BBa_C0161 enyme luxI (-LVA) BBa_C0070 Enzyme, rhlI (LVA+), Makes signaling molecule BBa_C0170 enzyme rhlI (-LVA) BBa_C0078 Enzyme, lasI (LVA+), Makes signaling molecule BBa_C0178 Enzyme lasI (-LVA) BBa_C0076 Enzyme, cinI (LVA+), Makes signaling molecule BBa_C0176 enzyme cinI (-LVA) BBa_C0083 Enzyme, aspA (LVA+), makes aspartate BBa_C0020 CheY, makes bugs tumble

SINGLE BLOCKS - STICKY TAGS

BBa_M0020 GST tag binds glutathione BBa_M0021 Poly-His tag binds nickel, zinc, cobalt affinity resins BBa_M0024 biotin tag – binds streptavidin BBa_M0025 MBP tag – binds maltose BBa_M0027 CBP tag (cellulose binding peptide) BBa_M0030 Poly-Arg binds cation resin BBa_M0031 Strep-tag II binds biotin

SINGLE BLOCKS - TAGS

DEGRADATION TAGS

BBa_M0040 - LVA, slow degradation BBa_M0042 - LAA, medium degradation BBa_M0044 - AAV, fast degradation BBa_M0040 - ASV, fastest degradation

=Introduction= Our brainstorming meeting on 3-16 gave us several preliminary ideas which we will explore via modeling and research Things I learned at the teach the teachers meeting

Standard plasmids: pSB1AK3 ("ampicillin kanamycin")

                  pSB1AT3 ("amplicillin tetracycline)
                  pSB1AC3 ("ampicillin chloramphenicol)

all of these are high copy number, if inappropriate, also provided are inducible plasmids:

                  pSB2K3 (IPTG inducible)

the "3" refers to the MCS and the transcriptional terminator We will be provided with a plate with dried DNA samples, we can amplify the ones that we want There are standard cloning protocols that use specific sites; products are checked by sequencing with promoter sequences VF ("verification forward") and VR ("verification reverse")

They often use 3-way ligations selected with 2 antibiotics to create composites You can put > 1 plasmid in a cell; chose according to copy number desired (eg, you might want GFP at high copy and AraC at low...)

EVERYONE should have a BioBrick account with their real name go here: http://partsregistry.org/Help:Create_a_Registry_Account It is important to document parts every time we create them They should have the "BioBrick ends" what does this mean?

We will get 2 384-well plates with the DNA; pierce the foil, dissolve in 30-50 microliters, and transform it's colour-coded with food coloring: KANAMYCIN = red; TET = yellow; CHLOR = green; AMP = orange the key to what's in each well will be provided on the registry fill out address on Group pages next week! when we send parts, they should be sent as stabs labeled w/ part name, plasmid, cell (on Help page) (or foreign schools can send dried DNA) as soon as a part is Available, it will be sent to EVERYONE, that way they don't worry about shopping lists The letters on the plates are very small, so watch out